General Histology & Molecular Techniques Flashcards
Explain Principles of Immunohistochemistry
Involves the process of selectively identifying antigens (proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
The indirect method involves an unlabeled primary antibody (first layer) that binds to the target antigen in the tissue and a labeled secondary antibody (second layer) that reacts with the primary antibody. As mentioned above, the secondary antibody must be raised against the IgG of the animal species in which the primary antibody has been raised. This method is more sensitive than direct detection strategies because of signal amplification due to the binding of several secondary antibodies to each primary antibody if the secondary antibody is conjugated to the fluorescent or enzyme reporter.
Explain how to perform a Gram stain
A Gram stain is a method of staining tissues, in order classify bacterial species into two large groups: gram-positive bacteria and gram-negative bacteria.
Based on differences in cell walls of bacteria (thickness of the peptidoglycan layer)
Method:
1) a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixation kills some bacteria but is mostly used to affix the bacteria to the slide so that they don’t rinse out during the staining procedure.
2) Addition of iodine, which binds to crystal violet and traps it in the cell
3) Rapid decolorization with ethanol or acetone
4) Counterstaining with safranin or carbol fuchsin
Ziehl–Neelsen staining is a type of acid-fast stain and is used to identify acid-fast organisms, mainly Mycobacteria.
Summary of acid-fast stain (Ziehl–Neelsen stain)
1) Primary dye - Carbol fuchsin
2) Decolorizer - Acid alcohol
3) Counter stain - Methylene blue/malachite green
Explain how you would validate a new IHC stain in your laboratory
- Work with your senior scientist who does the IHC
- Follow the manufacturers protocols / data sheet
- Antigen retieval relies on temperature, pH and set processing time
- Use of both positive and negative control tissue (either commerical or in house)
- Several practice runs to optimise antibody before putting into general use
Explain how DIFL works
Direct immunofluorescence is a technique for detecting deposits of immunoglobulins and complement proteins in biopsies of skin, kidney and other organs.
Primary, or direct, immunofluorescence uses a single antibody that is chemically linked to a fluorophore. The antibody recognizes the target molecule and binds to it, and the fluorophore it carries can be detected via microscopy.
Secondary, or indirect, immunofluorescence uses two antibodies; the unlabeled first (primary) antibody specifically binds the target molecule, and the secondary antibody, which carries the fluorophore, recognises the primary antibody and binds to it. Multiple secondary antibodies can bind a single primary antibody. This provides signal amplification by increasing the number of fluorophore molecules per antigen.
In skin: IgG, IgM, IgA, C3 and fibrin (frozen, perilesional)
How do you decalcify tissue?
Bone decalcification is the softening of bones due to the removal of calcium ions
There are two categories of decalcifying agents for removing calcium ions:
1) Chelating agents e.g Ethylenediaminetetraacetic acid (EDTA).
- chelating agents take up / absorb the calcium ions.
- slow but better preservation of cytologic detail, better for IPX and molecular tests
2) Acids -weak acids e.g 10% formic acid, strong acids e.g HCL
- acids help produce a solution of calcium ions
- fast but risk of losing cellular detail (esp. if over decalcify), can lose antigenicity for IPX and destroy DNA for molecular tests.
** ratio 20 : 1; change every 24 hours.
Explain principles of a DQ stain.
Diff-Quik is a commercial Romanowsky stain variant used to rapidly stain a variety of pathology specimens. Based on a modification of the Wright-Giemsa stain (faster).
Steps:
1: Fixative: 100% ethanol / methanol
2: Diff-Quik solution I (eosinophilic) Xanthene dye (Eosin Y), pH buffer
3: Diff-Quik solution II (basophilic) Thiazine dye, methylene blue, azure A, pH buffer
What special stains are commonly used in skin biopsies?
PAS - fungi, basement membrane
Other bug stains: Fite, Warthin-Starry, GMS, Gram
Hales - dermal mucin
Perls - iron deposition
Masson - collagen deposition, fibrosis, perforating disorders
EVG - elastin fibres
CR - amyloid deposition
What are the components of formalin in your lab
10% neutral buffered formalin is a 1:10 dilution of 100% formalin in water i.e. 1 part 100% formalin to 9 parts water with sodium phosphate added as a buffer.
100% formalin contains ~40% formaldehyde therefore a 1:10 dilution contains about 4% formaldehyde.
If in doubt check manufacture data sheet (by safety cabinet) or ask lead scientist.
What is in IMDM or RPMI?
IMDM and RPMI are synthetic cell culture media.
Like all cell culture media they contain nutrients to ensure cell survival ex vivo eg. amino acid, peptides, minerals, vitamins and buffering agents.
Exact details check manufacturers data sheet.
Explain Principles of FISH
Fluorescent in situ hybridisation (FISH)
Molecular cytogenetic technique that uses a fluorescent probe that binds specifically to part of a nucleic acid sequence.
Probe = single strand of DNA or RNA that is complementary to the region of interest.
Fluorecent microscope = used to identify where fluorescent probes are bound to chromosomes
Can be used to DNA, RNA, tissue (fresh frozen, FFPE) and circulating tumour DNA
5 STEPS
- DNA unmasking
- Probe and target denaturation
- Probe and target hybridisation
- Detection of target
- Image analysis under fluorescent microscope
Advantages of FISH
Relatively quick
Large number of cells can be scored over a short period
High sensitivity and specificity
Cytogenetic data can be derived from non dividing or terminally differentiated cells
Disadvantages of FISH
Relatively expensive
Need to know target of interest
Sample may not be suitable: degrade, necrotic
Cannot detect small mutations / rearrangements (low end of resolution ~ 15mb)
Fluorescence fades over few weeks - need to photograph!
Principles of Flow Cytometry
Flow cytometry is a technique for the rapid analysis of a statistically significant population of cells at a single cell level.
Usually a single cell suspension, labelled with fluorescent markers or antibodies and passed through a laser beam one cell at a time.
The light absorbed and emitted as wavelengths is can be sensed by photomultiplying tubes (PMT’s) and converted into data that is produced via computer.
What are Barr Bodies?
Barr bodies are an inactive X chromosome present in a cell which has more than one X chromosome.
Lyonisation = process in which a cell with multiple X chromosomes, all but one are inactivated during embryogenesis.
Therefore females with two X chromosomes will have 1 Barr body per somatic cell.
Males with one X chromosome will have 0 Barr bodies per somatic cell.
Can use Barr bodies to determine if tissue comes from a male or a female.
Common Uses for Flow Cytometry
Immunophenotyping lymphomas and leukaemias
Histocompatibility cross matching
Transplant rejection
Auto and alloimmune disorders
Reticulocyte enumeration
Feto maternal haemorrhage quantification
Advantages of Flow Cytometry
Can measure large number of parameters on the same sample
Fast - can collect information about a large number of cells very quickly
Cytospin taken at time of preparing sample can be used for morphologic assessment / checking concordance
Disadvantages of Flow Cytometry
Expensive
Sophisticated instrument, requires experienced and trained scientists to run and interpret results
Sample may not be representative
Explain Principles Of ISH
In Situ Hybridisation
A method for detecting nucleic acid sequences (RNA / DNA) in histologic section by applying a complementary strand of nucleic acid to which a reporter molecule is attached.
Types of Probes:
dsDNA, ssDNA, RNA, synthetic oligonucleotides
Labelling Techniques:
radioactive isotope, biotin, fluorescent dye
What is the Urovysion Test For?
FISH test using enumeration probes for the centromere regions of chromosomes 3, 7, 17 and probe for p16 (9q21).
Urothelial carcinoma is characterised by aneuplody with typically gains of chromosomes, 3, 7 and 17 and loss of p16.
“Positive” FISH result can also be seen in:
Primary bladder adenocarcinoma
SCC of the bladder
Prostate adenocarcinoma
Colorectal carcinoma
- > correlate wtih clinical, radiologic findings as well as urine cytology +/- histology
- > particularly given prostate cancer and colorectal cancer can secondarily involve the bladder
Explain PD1 / PDL1 Testing
The PD1 receptor and its ligands (PDL1 / PDL2) are part of a group of checkpoint inhibitors that act as coinhibitory factors in the cancer immunity cycle.
Tumour cells can hijack this process in order to evade detection by the immune system.
- > PDL1 is overexpressed by tumour cells
- > binds to PD1 on activated T-cells
- > leads to inhibition of cytotoxic T cells
Monoclonal antibodies against PDL1 and PD1 (eg pembrolizumab) can allow the immune system to recognise the tumour cells as non-self meaning cytotocic T cells and NK cells can attack tumour cells.
Detection of PDL1 expression by IPX determines if disease will be treatment responsive.
In NZ PDLI testing can be offered for NSCLC, TNBC, Urothelial ca, Gastric ca and H&N SCC.
Limitations of Small Biopsy Specimens
May not be representative of the lesion as a whole
Less material available for ancillary testing including molecular testing
Causes of False Positives in FISH Testing
Aneuploidy or polyploidy can give excess number of signals (particularly important with enumeration probes)
Thick sections or very cellular tumours will create overlap of tumour cell nuclei leading to increased number of cell signals being detected
Low level signals may be over interpreted as positive
Sampling bias
Causes of False Negatives in FISH Testing
Rearrangement not covered by probe - genes too close together, rearrangement is not within the size range of FISH testing (e.g small rearrangements inside genes)
Inadequate target retrieval due to over or under fixation, decalcification
Failure of hybridisation due to technical issue e.g pH or temperature
Explain Principles of PCR
PCR = polymerase chain reaction
lab technique for rapid production or amplification of millions to billions of copies of a specific segment of DNA which can then be studied in more detail
Involves using short synthetic DNA fragments called primers to select the segment of genome to be amplified
Then multiple rounds of DNA synthesis to amplify that sequence
Need: DNA, primers, TAQ polymerase, nucleotides and buffer mix in a PCR tube and a thermal cycler
Steps: Denaturing, annealing and extension
How Does the Oncofocus Panel Work?
For somatic mutations in BRAF, EGFR, KIT, KRAS, and NRAS genes
The OncoFOCUS Panel employs two multiplexed PCR reactions. Amplification is followed by a single base extension reaction. The extension products are dispensed onto a Chip Array and detected via mass spectrometry.
After the sample run, the OncoMutation Report provides the list of positive mutations identified in the samples.
Explain Mass Spectrometry
Mass spectrometry (MS) is a technique used to measure the mass-to-charge ratio of ions. The results are presented as a mass spectrum, a plot of intensity as a function of the mass-to-charge ratio.
Three steps:
1) Ionisation e.g. by bombarding substance with electrons to create positively charged fragments
2) Fragments then separated according to mass:charge ratio by exposing them to electomagnetic field
3) Ions then detected & sorted using an electron multiplier
When a specific combination of source, analyzer, and detector becomes conventional in practice, a compound acronym may arise to designate it succinctly. One example is MALDI-TOF, which refers to a combination of a matrix-assisted laser desorption/ionization source with a time-of-flight mass analyzer. (Used with Oncofocus).
Uses for EM
Electron microscopy is useful for:
Typing Glomerulonephritis.
Metabolic Inherited Disorders (Heart / Liver)
Peripheral neuropathy
Myopathies
Primary ciliary dyskinesia
-> Also rapid viral identification, help with tumour typing
Steps for Preparing EM
- Small 1mm cube of tissue
- Primary fixation in glutaraldehyde
- Secondary fixation in osmium tetroxide
- Rinse and dehydration steps (ethanol)
- Dry with liquid CO2
- Embed by infusing with resin and cut thin (70nM) sections with diamond blade or cleaved glass
- Post stain and image
What are these structures?
Birbeck granules - Langerhan cells
What does this picture show?
Primary ciliary dyskinesia
Circle and arrow = loss / reduction of dynein arms
What does this cardiac biopsy show?
Fabry disease! (dense lamellar bodies or Zebra bodies)
What does this renal biopsy show?
Amyloid!
Name this testicular tumour
Leydig cell tumour with Reinke crystals
Name this kidney tumour
Juxtaglomerular cell tumour
Rhomboid shaped pro-renin granules in cytoplasm
Pleural tumour in adult male
Malignant mesothelioma with microvilli (and tight junctions)
Adrenal tumour in a 2 year old
Neuroblastoma with dysmorphic synaptic vesicles containing neurosecretory granules
Difference between MMR testing versus MSI testing
MMR testing performed using immunohistochemistry looking for presence or absence of MMR proteins (MLH1, PMS, MSH2, MSH6)
MSI testing is performed by PCR looking at DNA for an increase in nucleotide repeats between tumour and patients normal tissue
Discordance?
- check the same sample was used for both, not a mix up
- low tumour volume in MSI sample (eg very mucinous)
- if MSI testing is positive but IHC negative this may be due to a MMR protein tht is present but non functional
How is MLH1 Promoter Hypermethylation Tested?
- DNA is extracted from tumor areas on unstained slides and treated with bisulfite, which converts unmethylated cytosine residues to uracil. Methylated cytosines are protected from conversion.
- Treated DNA is then amplified by real-time PCR. A 100 bp segment of the MLH1 promoter known to contain eight CpG dinucleotides and be subject to methylation is targeted.
- Temperature dissociation. The amplified product is detected with intercalating dye. The melt characteristics of the generated amplicons are determined by loss of fluorescence as the double-stranded molecule is heated and becomes single-stranded.
The temperature at which the amplicon dissociates is dependent upon the sequence, and indicates whether the sequence was methylated or unmethylated.
BRAF gene
BRAF is a proto-oncogene within the MAPK pathway
Following a mutation within the gene it becomes an oncogene and becomes constitutively active driving cell signalling pathways and cell proliferation
BRAF mutations seen in wide range of cancers: PTC, LCH, ECG, HCL, melanoma, lung, CRC
Presence of BRAF mutation means that BRAF inhibitor therapy may be an option for the patient
Also if a BRAF mutation is present then it is unlikely that TKI’s for upstream targets e.g EGFR will be effective
ERBB2 / HER2 / Neu Gene
ERBB2 / HER2 / Neu is a proto-oncogene which codes for the HER2 receptor tyrosine kinase (member of EGFR family)
Amplification or overexpression of the HER2 gene resulting in activation of multiple signalling pathways (MAPK, STAT, p13K) which promote cell proliferation and prevent apoptosis
Implicated in multiple cancers: breast, gastric, salivary duct carcinoma, ovarian, lung adenocarcinomas
Important molecular target for monoclonal antibodies such as trastuzumab (Herceptin)
EGFR
EGFR is a receptor tyrosine kinase involved in cell signalling pathways that regulate cell division and survival
Amplification and mutations in EGFR gene lead to a constitutively active tyrosine kinase receptor with sustained cell signalling and proliferation
EGFR is implicated in a number of tumours (e.g lung adenocarcinoma and high grade gliomas) and is targetable
Long term use of TKI’s lead to drug resistance e.g. EGFRT790M - identifying this means patient can switch to 3rd generation TKI
RAS genes
RAS - family of genes and proteins (KRAS / NRAS / HRAS) involved in the MAPK signalling pathway
Expressed in all cell lineages and mutations that activate RAS are found in up to 20% of all human cancers (CRC, pancreatic up to 90%, lung adenocarcinomas)
Control cell signalling and cell proliferation
If KRAS mutation is present then EGFR TKI’s will be ineffective (as KRAS is downstream)
MYC
MYC is a proto-oncogene that codes for transcription factors
If MYC rearranges (e.g MYC-IgH translocation in BL) or amplified (e.g. neuroblastoma, medulloblastoma) then transcription factors are produced at a higher rate
RB1 gene
RB1 gene (13q) codes for the RB1 protein which is a negative regulator of the cell cycle
RB1 protein prevents cell cycle progression from G1 to S phase
Germline mutation = retinoblastoma (double hit) and osteosarcoma
Somatic mutations = spindle cell lipoma as well as some common cancers (lung, PDAC, breast)
Hijacked and disarmed by E7 oncoprotein following infection by HR HPV
VHL gene
VHL is a tumour suppressor gene which encodes a multifactorial protein that negatively regulates HIF
Germline mutations = VHL syndrome
Associated tumours: ccRCC, haemangioblastoma, phaeo, endolymphatic sac tumour, epididymal papillary cystadenoma, serous cystadenoma of pancrease, PanNET’s with clear cells
APC gene
APC is a tumour suppressor gene that codes for the APC protein which is a negative regulator of the cell cycle
APC controls ß-catenin concentrations and interacts with e-cadherin as part of Wnt pathway
Germline loss = FAP syndrome, attenuated FAP, Gardner, Turcot
Somatic mutation = colorectal cancer
TP53 gene
TP53 gene codes ofr p53 protein which induces cell cycle arrest, DNA repair and apoptosis.
“guardian of the genome” - mutations of p53 implicated in 1/2 - 1/3 all human cancers
Li Fraumeni - germline loss of one TP53 gene (high lifetime risk of breast, GBM, osteosarcoma, soft tissue sarcomas, adrenal cortical carcinoma)
p53 hijacked by E6 oncoprotein in hr HPV infection
BRCA2
BRCA2 gene codes of BRCA2 protein which mediates the orderly assembly of DNA repair proteins
Germline mutation: BRCA2 = hereditary breast and ovarian cancer
Breast cancer, including male
Ovarian cancer, especially serous
Prostate
Pancreas
Sporadic: Breast cancer and NSCLC
CDKN2a
CDKN2A is a gene ubiquitously expressed in many tissues and cells. The gene codes for two proteins p16 and p14arf.
p16 and p14arf act as tumor suppressors by regulating the cell cycle.
p16 activates the retinoblastoma (Rb) family of proteins, which block traversal from G1 to S-phase. p14ARF activates the p53 tumor suppressor
Somatic mutations common in many cancers including melanoma, mesothelioma and high grade gliomas.
Germline mutations of CDKN2A are associated with familial melanoma, glioblastoma and pancreatic cancer
NF1 gene
NF1 gene codes for the neurofibromin protein, a GTPase activating protein that negatively regulates the RAS / MAPK pathway
NF1 is the most common single gene disorder in humans. AD but half of cases arise sporadically, phenotypic variability
NF1 - diffuse and plexifrom neurofibromas, multiple NF’s, cafe au lait spots, Lisch nodules in iris, PA of optic nerve
Sporadic mutations seen in GIST, melanoma, lung and GBM
Interpret Gene Sequencing Data / Electropherogram
Typical electropherogram covers a single PCR product (average # of nucleotides 200 - 600) depending on size of gene being sequenced
Comparative analysis of patient sample and a DNA sample without a mutation
Each peak represents a single nucleotide in the DNA sequence.
Each nucleotide has a differnt colour (A = green, T=red. C=blue, G= black)
Sequencing analysis usually performed by software
Comparative Genomic Hybridisation
Molcular cytogenetic technique for analysing copy number variations relative to ploidy level in the DNA of a test sample compared with a reference sample
Aim to compare 2 genomic DNA samples from 2 sources because they are suspected to have differences in gains or losses of chromosomes / subchromosomes
Array CGH with competitive FISH has a resolution of 5 - 10 kilobases
Next Generation Sequencing
Sequencing of multiple specimens in parallel
Targeted gene panel, whole exome, or whole genome
Multiple commercially available platforms with varying engineering configurations and forms of sequencing chemistry
Basic Steps:
- Build library by extracting DNA and amplification by PCR
- Sequencing
- Data extraction and bioinformatics
